Under physiologic conditions, binding of insulin to the insulin receptor stimulates its intrinsic tyrosine kinase activity, leading to tyrosine phosphorylation of the insulin receptor substrates (IRS1 and IRS2) (Virkamaki et al., J. Clin. Invest. 103:931–943, 1999; White, Mol. Cell. Biochem. 182:3–11, 1998), and activation of downstream signaling pathways via the recruitment of cytoplasmic effector proteins containing SH2 domains which recognize tyrosine phosphorylated IRS proteins. For example, recruitment of the p85 subunit of the PI3K (Ruderman et al., Proc. Natl. Acad. Sci. USA 87:1411–1415, 1990) in response to insulin triggers a phospholipid dependent kinase cascade that culminates in the activation of a protein kinase B (PKB)/Akt (Brazil and Hemmings, Trends Biochem. Sci. 26:657–664, 2001). The activation of PI3K appears particularly critical for insulin signaling; most of the effects of insulin on glycogen synthesis and glucose transport are blocked in cells treated with PI3K inhibitors (Shepherd et al., Biochem. J. 305:25–28, 1995).
Diabetes mellitus is among the most common of all metabolic disorders, affecting up to 11% of the population by age 70. Type I diabetes (also referred to as insulin dependent diabetes mellitus or IDDM) represents about 5 to 10% of this group and is the result of a progressive autoimmune destruction of the pancreatic beta-cells with subsequent insulin deficiency.
There are two classes of type II diabetes (also referred to as non-insulin dependent diabetes mellitus or NIDDM). One typically presents in older people; thus it is sometimes called mature onset diabetes. Another form, though similar to mature onset, presents in a subject at a very early age. Type II diabetes represents 90–95% of the affected population, more than 100 million people worldwide (King and Zimmer, Wld. Hlth. Statist. Quart. 41:190–196, 1988; Harris et al., Diabetes Care 15:815–819, 1992), and is associated with peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion (DeFronzo, Diabetes 37:667–687, 1988). Family studies point to a major genetic component (Newman et al., Diabetologia 30:763–768, 1987; Kobberling, Diabetologia 7:46–49, 1971; Cook, Diabetologia 37:1231–1240, 1994). However, few susceptibility genes have been identified.
Type II diabetes is characterized by a patient's inability to respond to insulin and/or insufficient insulin secretion. Insulin exerts a dominant effect on the regulation of glucose homeostasis. In the liver, insulin inhibits the production of glucose by inhibiting gluconeogenesis and glycogenolysis. Insulin is thought to act by causing cells to absorb glucose from the blood stream. Once absorbed, the liver converts glucose to glycogen. The liver supplies glucose by converting glycogen stores to glucose. Insulin also has a major role in the regulation of protein and lipid metabolism through a variety of actions that affect the flux of protein and lipid substrates.
Key molecules in the regulation of insulin are the protein kinase B/Akt family of enzymes. The protein kinase B (PKB)/Akt kinases consists of at least three members (Akt1, Akt2, Akt3), collectively referred to herein as “Akt”, that share extensive sequence homology but appear to perform distinct biological functions (Brazil and Hemmings, supra). Targeted disruption of Akt2, for example, leads to insulin resistance and glucose intolerance due to elevated hepatic gluconeogenesis and reduced glucose uptake in skeletal muscle (Cho et al., Science 292:1728–1731, 2001). By contrast, disruption of the Akt1 gene leads to growth retardation and apoptosis in certain tissues, with no apparent change in glucose homeostasis (Chen et al., Genes Dev. 15:2203–2208, 2001; Cho et al., J. Biol. Chem. 276:38349–38352, 2001). The mechanism underlying functional specification of Akt1 and Akt2 in liver and other tissues is unclear, but may involve subtle differences in substrate preference or in the ability of these kinases to associate preferentially with certain modulatory factors. In a recent study, Hemmings and coworkers described a C-terminal Akt modulatory protein, referred to as CTMP, that inhibits Akt activity at the plasma membrane and in the cytoplasm by binding to the C-terminal regulatory domain of Akt and blocking its phosphorylation at Thr308 and Ser473 (Maira et al., Science 294:374–380, 2001). Relative affinities of CTMP for Akt1 or Akt2 were not examined in this study, however.
Following activation in response to insulin, Akt inhibits glycogenolysis and promotes glycogen synthesis via direct phosphorylation of glycogen synthase kinase 3β at Ser9 (Brady et al., J. Biol. Chem. 273:14063–14066, 1998; Delcommenne et al., Proc. Natl. Acad. Sci. USA 95:11211–11216, 1998; Hajduch et al., Diabetes 47:1006–1013, 1998; Mitsuuchi et al., J. Cell. Biochem. 70:433–443, 1998; van Weeren, J. Biol. Chem. 273:13150–13156, 1998). Akt also appears to block gluconeogenic genes such as glucose-6-phosphatase and PEPCK, in part by phosphorylating and promoting nuclear export of members of the Forkhead family of transcriptional activators (Guo et al., J. Biol. Chem. 274:17184–17192, 1999; Kops et al., Nature 398:630–634, 1999; Nakae et al., J. Biol. Chem. 274:15982–15985, 1999). In recent studies, insulin has also been found to block gluconeogenesis by inhibiting the expression of the nuclear hormone coactivator PGC-1, although the underlying mechanism has not been elucidated (Herzig et al., Nature 413:179–183, 2001; Yoon et al., Nature 413:131–138, 2001).
In response to insulin stimulation, Akt is recruited to the plasma membrane via an interaction between its pleckstrin homology (PH) domain and phosphoinositol-(3, 4, 5)P3 (PI3P), a product of phosphoinositol 1,3-dependent kinase (PI3K). Binding to PI3P is thought to promote a conformational change in Akt that renders the protein competent for subsequent activation events. Following binding to PI3P, Akt is phosphorylated at two residues: Thr308 within the active loop and Ser473 in the regulatory domain (Brazil and Hemmings, supra). Thr308 phosphorylation is mediated by the phosphoinositide-dependent kinase-1 (PDK-1) a PH domain kinase whose activity is also regulated by PI3K. Consistent with the importance of Thr308 phosphorylation for Akt catalytic activity, Akt activation is absent in PDK1−/−cells (Williams et al., Curr. Biol. 10:439–448, 2000). Ser 473 phosphorylation also contributes to Akt activation, although the identity of the Ser 473 Akt kinase is unknown.
By contrast with the well-characterized events leading to Akt activation, the mechanisms by which Akt activity is attenuated following insulin stimulation are less clear. Nevertheless, a number of upstream negative regulators have been identified; and the best characterized to date is the phosphatase-tensin homolog protein (PTEN), a potent lipid phosphatase that blocks Akt activation by dephosphorylating 3-phosphoinositides. PTEN is often mutated in a variety of sporadic cancers as well as in certain hamartoma syndromes; and tumors that harbor inactive PTEN correspondingly often contain elevated levels of Akt activity (Backman et al., Nat. Genet. 29:396–403, 2001; Ramaswamy et al., Proc. Natl. Acad. Sci. USA 96:2110–2115, 1999). In addition to PTEN, the SH2-containing inositol 5′ phosphatase (SHIP), which hydrolyzes PI(3,4,5)P3 to PI(3,4)P2, has also been found to inhibit Akt activity; and SHIP −/−cells exhibit prolonged activation of Akt upon stimulation (Aman et al., J. Biol. Chem. 273:33922–33928, 1998; Liu et al., Genes Dev. 13:786–791, 1999).
Although lipid phosphatases constitute important upstream regulators of Akt, the Ser/Thr protein phosphatase 2A (PP2A) also appears to inhibit Akt via direct dephosphorylation of Thr308 and Ser473. In this regard, dephosphorylation and inactivation of Akt in response to hyperosmotic shock can be blocked by addition of calyculin, a relatively specific inhibitor of PP2A (Meier et al., EMBO J. 17:7294–7303, 1998). The degree to which PP2A contributes to Akt inactivation in vivo, however, is not well understood.
In the fed state, insulin promotes glucose homeostasis by stimulating glucose uptake in muscle and fat, and by blocking glucose production in liver (Saltiel and Kahn, Nature 414:799–806, 2001). Mice with an insulin receptor knockout in liver show glucose intolerance due in part to elevated glucose production (Michael et al., Mol. Cell. 6:87–97, 2000). Unchecked hepatic gluconeogenesis is an important contributor to fasting hyperglycemia in Type II diabetes, suggesting that the liver is a major site for glucose intolerance and insulin resistance in this disease.
In the fasted state, blood glucose levels are maintained through hepatic output of glucose, mediated predominantly by a fall in insulin and a rise in counter-regulatory hormones, i.e. glucagon (cAMP) and adrenal glucocorticoids. Glucagon promotes gluconeogenesis, in part, by stimulating the protein kinase A (PKA) mediated phosphorylation of the cAMP responsive element binding protein (CREB) (Imai et al., J. Biol. Chem. 268:5353–5356, 1993; Liu et al., J. Biol. Chem. 266:19095–19102, 1991; Quinn and Granner, Mol. Cell. Biol. 10:3357–3364, 1990). Expression of a dominant negative CREB inhibitor, referred to as A-CREB, in liver either acutely by infection with A-CREB Adenovirus, or chronically by transgenic expression in mice, causes hypoglycemia with reduced expression of all gluconeogenic genes (Herzig et al., supra). CREB was found to promote expression of the gluconeogenic program by stimulating expression of the nuclear hormone receptor coactivator PGC-1 (Herzig et al., supra; Yoon et al., supra) via a cAMP response element (CRE) site in the PGC-1 promoter. The ability of PGC-1 to promote expression of gluconeogenic genes in response to glucocorticoid signals likely explains the cooperativity between cAMP and glucocorticoid pathways in regulation of hepatic glucose production (Herzig et al., supra; Yoon et al., supra).
In addition to stimulating glucose output, chronic fasting has been found to induce insulin resistance in liver downstream of the insulin receptor. Indeed, glucocorticoids and, to a lesser extent, catecholamines, also induce hepatic insulin resistance by blocking post-receptor insulin signaling (Paez-Espinosa et al., Mol. Cell. Endocrinol. 156:121–129, 1999; Rao, Metabolism 44:817–824, 1995). The mechanism underlying insulin resistance in this setting remains obscure, but suggests the presence of an inducible negative signal that impairs insulin signaling under fasting conditions.
Thus, there remains a need in the art for methods to modulate enzymatic pathways in glucose regulation, particularly modulators of PKB/Akt kinases. Among other essential pathways in glucose regulation, these critical kinases inhibit glycogenolysis, promote glycogen synthesis, and block gluconeogenic genes. These pathways are all involved in diabetes mellitus, and modulators of PKB/Akt kinases present novel methods of diagnosis and treatment.